Hyclone rpmi 1640 medium

EC109 and KYSE180 cells were cultured in HyClone RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS at 37 °C in a humidified incubator containing with 5% CO₂. HEK293T cells were cultured in DMEM high glucose medium (Biowest) supplemented with 10% FBS at 37 °C in a humidified incubator containing with 5% CO₂.

Human hepatocytes (L02 cells) were obtained from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in HyClone RPMI1640 medium (Thermo Scientific, Beijing, China) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere at 5% CO₂. Cells of 3–5 passages were used in our study. Hyp was dissolved in dimethylsulfoxide (DMSO) as a stock solution and diluted with the culture medium before cell culture treatment, while control cells were treated with equal amounts of DMSO at a final concentration of < 0.1%.
The dose of Hyp and morin was selected according to previous publications (Zhu et al., 2012 (link); Madankumar et al., 2014 (link); Li et al., 2018 (link)). To assess the effects of Hyp on expression of PHLPP and related proteins, liver cells were treated with morin (10 μM for 3 h) or Hyp (100 μM for 1, 3, or 6 h), while to assay the effect of Hyp on t-BHP-induced protein expression, cells were treated with morin (10 μM) and Hyp (100 μM) for 3 h and 6 h, respectively and then with t-BHP (250 μM for 90 min). Thereafter, the cells were collected for the following experiments.

Thp1 cells were cultured at 37°C with 5% carbon dioxide in HyClone RPMI 1640 medium modified (Thermo Fisher Scientific Inc.), 10% fetal bovine serum (FBS) (Gibco, Life Technologies Corporation), and 100 mg/mL streptomycin (HyClone). TCP was sterilized and placed into 24-well plates (100 mg/well) using a small spatula and then into cultures with 500 μL/well of Thp1 culture medium. After 24 h of incubation, the culture medium was collected as the TCP extract.
Thp1 was first induced to macrophages by culture with 100 ng/mL of phorbol myristate acetate (PMA) (Sigma) at a density of 1 × 10⁶ cells/mL for 48 h. Then the culture medium was changed to culture medium containing TCP extract, 500 ng/mL lipopolysaccharide (LPS) (PeproTech), and 20 ng/mL human interleukin-4 (IL-4) (PeproTech). After incubation for 1 h, the supernatants of Thp1 cells cultured with TCP extract were collected.
Human bone marrow stem cells (HBMSCs) were obtained from patients undergoing iliac bone graft with informed written consent. The procedure was approved by the Ethics Committee at Wuhan University, China. HBMSCs were cultured in a-MEM medium (Thermo Fisher Scientific Inc.) at 37°C with 5% carbon dioxide.

The H358, H460, NCIH23, SKLU-1, SW960, A427, H727 (KRAS^Mut), H1650 (KRAS and EGFR double mutant), H1975, HCC827, HCC2279 (EGFR^Mut), HCC1666, H322M, H522, Calu-3, H1299 (KRAS and EGFR wild type), BEAS-2B (nontumorigenic lung epithelial cells), CCD-18Lu (nontumorigenic lung fibroblasts), and human embryonic kidney (HEK) 293 T cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The GFP-positive KRAS^G12C mutant stable cell lines H1299 KRAS^G12C_K104, H1299 KRAS^G12C_K104R, and H1299^H363Y were generated by stably cotransfecting H1299 KRAS^WT cells. The cells were cultured in either HyClone RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) or Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA), each containing 10% FBS and 1% penicillin and streptomycin. Cells were maintained in a 37 °C humidified incubator with 5% CO₂ in air. All cell lines were authenticated by short tandem repeat (STR) analysis and were free of mycoplasma contamination.

Human IL-1β (Cat: EHC002b) and TNFα (Cat: EHC103a) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Beijing NeoBioscience Technology Co., Ltd., China. Hyclone RPMI 1640 medium (SH30809.01B) was from Thermo Fisher scientific Co., Ltd. (Beijing, China). FBS was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). Uric acid sodium salt (U2875) and phorbol 12-myristate 13-acetate (PMA) (product number: 79346) were ordered from SIGMA-ALDRICH, Co. (St. Louis, USA); Nikon Microimaging System (TE2000-U, Tokyo, Japan); Microplate reader (BioTek Synergy2, Vermont, USA); Inverted microscope (CKX-31, Olympus Corporation, Tokyo, Japan); CO₂ incubator (New Brunswick Scientific Co., Inc., New Jersey, USA); Esco Airstream Class II Biological Safety Cabinet (Beijing, China); Transmission electron microscope (FEI Tecnai G²12, Holland); Rotavapor (BUCHI, Flawil, Switzerland).